Retinol dehydrogenase 10 reduction mediated retinol metabolism disorder promotes diabetic cardiomyopathy in male mice

Diabetic cardiomyopathy is a primary myocardial injury induced by diabetes with complex pathogenesis. In this study, we identify disordered cardiac retinol metabolism in type 2 diabetic male mice and patients characterized by retinol overload, all-trans retinoic acid deficiency. By supplementing type 2 diabetic male mice with retinol or all-trans retinoic acid, we demonstrate that both cardiac retinol overload and all-trans retinoic acid deficiency promote diabetic cardiomyopathy. Mechanistically, by constructing cardiomyocyte-specific conditional retinol dehydrogenase 10-knockout male mice and overexpressing retinol dehydrogenase 10 in male type 2 diabetic mice via adeno-associated virus, we verify that the reduction in cardiac retinol dehydrogenase 10 is the initiating factor for cardiac retinol metabolism disorder and results in diabetic cardiomyopathy through lipotoxicity and ferroptosis. Therefore, we suggest that the reduction of cardiac retinol dehydrogenase 10 and its mediated disorder of cardiac retinol metabolism is a new mechanism underlying diabetic cardiomyopathy.

RDH10 in the hearts of db/db mice via adeno-associated virus 9 -RDH10 injection. A nearly perfect match to RDH10 levels in control mice was achieved. How did the authors determine the dose of the virus that gave such a perfect match to control levels? Have they tried a higher dose to increase RDH10 amount further? Did the amount of RDH10 in the heart vary depending on the viral dose? The authors report that pigment epithelium-derived factor (PEDF) binds to RDH10. Considering that RDH10 is ER-bound whereas PEDF is a secretable soluble protein, this finding is surprising and needs to be substantiated by additional experimental evidence. Calorie restriction was reported to alleviate diabetic cardiomyopathy. Have the authors tried this approach to see if RDH10 levels go up? Overall, this study has a potential to make an important contribution to the field if the aforementioned concerns are addressed appropriately. English needs editing.
Reviewer #2 (Remarks to the Author): This study by Wu and colleagues studied the effects of retinol dehydrogenase 10 (RDH10) reduction in diabetic heart as a causative factor for diabetic cardiomyopathy. They show increased cardiac retinol content in the diabetic heart, which is associated with reduced retinoic acid due to the downregulation of the key rate-limiting enzyme RDH10. This is an interesting and novel finding in relation to diabetes and the authors have done a series of experiments to confirm their hypothesis. The authors have provided the full analysis and all blots. My comments are as follows 1. The number of human samples are too small to provide a conclusion or translation value of the study. Importantly, it is not clear on the duration of diabetes in these individuals and whether they were normalized for the intake of medicine and other associated comorbidities. For example, are these patients prescribed any multivitamins? used to conclude the results is very limited. 2. Can the authors please confirm how many times the molecular analysis was repeated independently? 3. Several previous studies have shown the development of systolic and diastolic dysfunction, fibrosis and apoptosis in the db/db mice starting from 20-24 weeks of age. However, in this study, the animals appear to be healthy at least until 24 weeks of age. Please explain the reason for this discrepancy. 4. In line with this, Supplemental figures 2C and 2D show a significant difference between db/db and db/m at 24 weeks of age in EF and FS. However, Figure 2C, where the animals were treated with Rol shows no difference in EF and FS. Not clear why this is different here. Same in Figure 3 experiments with atRA 5. Similarly, why did the authors choose to follow up to 28 weeks for experiments with atRA. They should have kept the time points consistently across different treatments to make it easy for comparison. 6. Further, no data is presented for 24 weeks time point for the study where they overexpressed RDH10.
Reviewer #3 (Remarks to the Author): In this study, the authors showed that diabetic cardiomyopathy is associated with increased levels of retinol (Rol), decreased levels of all-trans retinoic acid (atRA), and decreased expression of RDH10 (a key enzyme that mediates the conversion of Rol to atRA). Using overexpression and conditional KO mouse models as well as Rol/atRA supplementation in mice, the authors further showed that dysregulation of Rol metabolism indeed plays a causal role in diabetic cardiomyopathy. They also proposed that deregulation of Rol metabolism affects diabetic cardiomyopathy through several mechanisms, including inducing lipotoxicity and ferroptosis.
1. Fig. 2: the authors claim that Rol overload promotes myocardial injury in T2DM mice. However, compared to db/db mice, db/db+Rol mice did not show much difference in terms of heart size and heart/tibia ratio (Fig. 2E), and cardiomyocyte area (Fig. 2G). Therefore, it seems that the data does not justify their conclusion. Note that in these figures, the statistical analysis (which shows the difference is statistically significant) was conducted between db/m and db/db+Rol groups, but this is wrong. Only the comparison between db/db and db/db+Rol groups would make sense here.
2. page 10 "To further identify the regulator of cardiac RDH10 in the heart of T2DM, we treated neonatal mouse primary cardiomyocytes (NMPCs) with high glucose, palmitic acid (PA), recombinant leptin or recombinant pigment epithelium-derived factor (PEDF)". To guide broad readers better here, the authors need to provide a brief justification on why they treated in mice, for example, because these are known factors involved in T2DM? Likewise, "PEDF, which has been shown to be associated with metabolic diseases 15, 16, 17, is reduced and promotes myocardial injury in the heart in T2DM (unpublished observations)" do the authors mean that the reduction of PEDF expression promotes myocardial injury in the heart in T2DM? The authors need to modify this sentence to make it more clarified. 4. There is a significant disconnection between Fig. S5 and all other figures in this manuscript. PEDF was never mentioned again in the rest of the manuscript, so what is the point to study PEDF in this study? Considering the issues associated with PEDF data (see points 2 and 3) and the lack of data showing that PEDF is important in T2DM (mentioned as unpublished observations. Many journals now do not allow this statement, such that the authors should either show the data or not make the relevant point), I suggest the authors remove Fig. S5 and related text in manuscript. This would not affect the conclusion of this study. 7. One weakness of this study is that how atRA deficiency causes GPX4 expression suppression, iron accumulation, and CD36-mediated FFA uptake to promote lipotoxicity (as summarized in Fig. 9) remains unknown. Some minimal mechanistic studies can further strengthen the study.
Minor points: 1. The data in Fig. 6J need to be presented with quantification (from at least three data sets) and statistical analyses.
2. Fig. 7A-D, the text states that 4-HNE levels were reduced in RDH10-cKO mice, but the data showed opposite results.
3. Also, the authors mentioned that MDA, iron and non-heme iron levels were not changed in RDH10-cKO mice. Even though ferroptosis is a form of iron-dependent cell death, iron levels per se are not good markers for ferroptosis, so it is not surprising that iron levels did not change here. However, MDA and 4HNE levels typically correlate well with ferroptosis in vivo. Fig 7B indeed shows that MDA levels were increased in RDH10-cKO mice, but the difference is not statistically significant probably because of the huge variation in the analysis. The authors should increase their sample size in their analysis. the upper part of the blot is cut off very close to RDH10 band and molecular weight 1 markers are not indicated on the images. To validate the identity of the band as RDH10, 2 the authors need to provide a clean full size image of the blot with a positive control 3 (recombinant RDH10) to show the size of the RDH10 protein, and include a negative 4 control (sample of their conditional KO total extract) side by side with samples from 5 db/db mice on the same gel/blot. Otherwise, there is a strong possibility that the authors 6 are detecting a non-specific band. 7 RESPONSE: Thank you for your valuable suggestion. We know that your main concern is the 8 specificity of the RDH10 antibody we used. The RDH10 antibody we used is the same as the 9 one in reference 32 1 ; the difference is that in western blotting experiments, we used the 10 concentration of 1:1000, which is recommended in the manual for this antibody, while the 11 concentration used in reference 32 was 1:3000. 12 To further verify the specificity of this antibody, we first followed your suggestion and used 13 RDH10 recombinant protein as a positive control to revalidate RDH10 expression in db/db and 14 3 manuscript, RDH10 expression was significantly downregulated in the hearts of 24-and 32-1 week-old db/db mice. 2 Second, we compared RDH10 expression in liver microsomes and heart microsomes in fed and 3 16-h-fasted mice. As shown in the image below, the expression of RDH10 decreased only in 4 the liver, not in the heart. It is well known that the liver is the major metabolic organ and is 5 sensitive to metabolic changes, whereas the heart is not as sensitive. As evidence, heart injury 6 always occurs at the latest stage of metabolic disorders. Therefore, we suggest that starvation 7 for 16 h or less does not lead to a decrease in RDH10 expression in the hearts of mice. showed larger or full size blot images of RDH10 in the source data file to prove the specificity 11 of our antibody and the reliability of our results. 12

In supplementary Fig 3, the same blot that was incubated with RDH10 antibodies needs 13
to be re-incubated with HSP90 antibodies and the full-sized image of the whole blot with 14 two protein bands corresponding to RDH10 and HSP90 on the same blot should be shown 1 to confirm the equal loading. Again, the size markers need to be included on all blots. 2 RESPONSE: Thank you for your valuable advice. First, it needs to be explained that in our 3 original manuscript, the strips of RDH10 and HSP90 were cropped from the same PVDF 4 membrane, and there was no difference in the loading. However, in view of the reviewer's query, 5 we revalidated our results of RDH10 western blotting in Supplementary Figure 4 a and hope  6 we have alleviated your doubts and can obtain your approval. 7 8 3. Similarly, the IHC results using RDH10 antibodies need to be validated using tissues 9 from RDH10 CKO mice. In general, IHC images are too small, and why is the background 10 blue for IHC in some images (T2D patient in Fig. S2D) but not in others? 11 RESPONSE: We know this comment reflects the same concern as that for the specificity of 12 our RDH10 antibody. We have followed your suggestions to verify the specificity of the 13 antibody we used, as described in the 2 responses above, and we hope our measures will address 14 your concerns. In addition, we performed IHC staining of RDH10 in RDH10-cKO mice, and 15 as shown in Supplementary Figure 4 b, cardiac RDH10 expression was decreased in RDH10-16 cKO mice, which is consistent with our western blotting and IF staining results. 17 The background of IHC staining was determined by the time of hematoxylin staining and the 18 9 levels? Have they examined the levels and activity of LRAT, the enzyme that esterifies 1 retinol? 2 RESPONSE: Thank you for your knowledgeable advice. Retinol administration by oral gavage 3 is common in related studies 3, 4 , and the dose of Rol we used was converted from the highest 4 dose that humans can use as a supplement without toxicity. We have previously attempted to 5 use higher doses, but they produced significant toxic effects in mice. 6 Numerous studies have found that dietary supplementation with Rol or its prerequisites can 7 significantly elevate Rol concentrations in the blood, liver, lungs and kidneys of mice, ducks 8 and humans 5, 6, 7 . However, we did not find any study suggesting that long-term Rol 9 supplementation does not alter serum holo-RBP4 levels. It is well known that skin toxicity and 10 neurotoxicity, as well as very significant teratogenic effects, occur if Rol is supplemented in 11 excess 8 . We believe that these phenomena cannot be caused solely by excessive accumulation 12 of Rol in the liver. 13 Due to the lack of a validated assay for blood holo-RBP4 levels in mice and to verify that Rol 14 supplementation elevates Rol levels in serum and other non-liver tissues, we measured the 15

RBP4 levels in serum and Rol levels in both the serum and hearts of db/db mice supplied with 16
Rol for 2 months (800 IU/2 days). Our results showed that supplementation with Rol over a 17 relatively short period significantly increased cardiac Rol levels but did not change serum RBP4 18 and Rol levels in these mice. This result surprised us, and it also made us realize that there are 19 still many unanswered questions in the field of retinol metabolism. We are unable to explain 20 this phenomenon, but we believe that through concerted efforts, researchers will be able to 1 solve the mysteries of retinol metabolism. 2 Because of Covid-19, we were unable to obtain a suitable antibody to measure the protein 3 expression level of LRAT within the revision period, we only measured the concentration of 4 cardiac retinyl esters, which were not altered in either db/db or db/db+Rol mice as shown in intraperitonealy into db/db mice at 5 µg/g body weight, which amounts to 250 µg/~50 g 8 db/db mouse weight daily for 28 weeks. This dose appeared to restore the atRA in db/db 9 heart to nearly perfectly matched levels to those in control mice. How did the authors 10 determine the exact dose to achieve such a perfect match? 11 11 RESPONSE: The way we provided atRA to db/db mice and the dose used were selected by 1 referring to a relevant study 9 . In the study we referenced, atRA served the purpose of treating 2 myocardial injury, but perhaps due to the difficulty of atRA measurement, the authors did not 3 measure the cardiac atRA levels in the mice before and after atRA supplementation. Due to the 4 extremely unstable physicochemical properties of atRA, measurement of atRA levels in tissue 5 is very difficult, so we specifically found a partner to help us measure cardiac atRA levels by 6 establishing a very reliable method. We did not anticipate that such a dose of atRA could so 7 perfectly restore the cardiac atRA content in db/db mice to about the same level as in db/m mice, 8 but it did. We have attached the original peak plots of the cardiac atRA content we performed 9 for your review and hope they will meet with your approval. 10 9. Notably, the size of the heart did not appear to change by much based on the images. Have they tried a higher dose to increase RDH10 amount further? Did the amount of 5 RDH10 in the heart vary depending on the viral dose? 6 RESPONSE: Thank you for your comments. In fact, we performed preliminary experiments 7 using three concentrations of AAV9 virus, 0.5*10^11, 0.8*10^11 and 1.2*10^11. We found 8 that 0.5*10^11 of AAV9 virus did not achieve the desired overexpression effect, while 9 1.2*10^12 of AAV9 virus caused the mice to die one after another in one month (including in 10 the AAV9-RDH10 group and AAV9-GFP group), which we think may have been caused by 11 the toxicity of AAV9 itself. 12 In addition, the aim of our experiment was to restore cardiac RDH10 expression in db/db mice 13 to the level in db/m mice rather than to achieve infinite overexpression of cardiac RDH10 in 14 db/db mice, so we ultimately chose a virus dose of 0.8*10^11. As shown in the figure below, 15 there were individual differences among different mice, so we could not accurately restore 16 cardiac RDH10 of each mouse to a level comparable to that in db/m mice. In Supplementary 13 Figure 5, we show the mice from the AAV9-RDH10 group with the cardiac RDH10 levels 1 closest to those of db/m mice. 2 11. The authors report that pigment epithelium-derived factor (PEDF) binds to RDH10. 3

Considering that RDH10 is ER-bound whereas PEDF is a secretable soluble protein, this 4
finding is surprising and needs to be substantiated by additional experimental evidence. 5 RESPONSE: Thank you for your comment. PEDF is a secretable protein that was once thought 6 to be produced only in the liver and adipose tissue, but as research has progressed, an increasing 7 number of researchers have found that cardiomyocytes can also produce PEDF 10, 11, 12, 13 . We 8 tried to verify the possibility that RDH10 binds to PEDF in cardiomyocytes using 9 immunofluorescence colocalization, but since we were not able to find an antibody that could 10 be used for mouse heart immunofluorescence staining for PEDF, we could only demonstrate 11 that PEDF was indeed present in the mouse myocardium by IHC staining and that PEDF 12 expression was significantly reduced in the hearts of db/db mice. 13 Human heart samples are very difficult to obtain, and this is a common challenge for researchers 13 in the field. However, to further increase the reliability of our study, we have done our best to 14 collect additional human samples and have updated our relevant results in Figure 1 h. We have 15 also summarized and presented information about these samples to the extent possible in 16 Supplementary Table 3, but unfortunately, we were unable to obtain information on whether 17 these patients had taken a multivitamin. shown different times of heart systolic dysfunction in db/db mice 14, 15, 16, 17 . Our group has also 10 made long-term observations on the heart function of db/db mice and found that heart systolic 11 dysfunction does not appear in db/db mice until 32 weeks of age. The relevant data were 12 published as a cover article in Theranostics in 2022 16 .  Figure 3. In addition, during this study period, we acquired the latest small animal ultrasound 21 detector, a Vevo 3100, by ourselves; only the data in Supplementary Figure 2 were detected 1 using a slightly earlier version of small animal ultrasound detector, the Vevo 2100, from others 2 before we acquired this instrument. We believe that different batches of mice and the different 3 small animal ultrasound instruments may have contributed to the differences in Supplementary 4 Figure 2 and others. However, our results in the same figure are based on data from the same 5 batch of animals with the same animal ultrasound detector, so the above differences should not 6 affect the reliability of our results. 7 5. Similarly, why did the authors choose to follow up to 28 weeks for experiments with 8 atRA. They should have kept the time points consistently across different treatments to 9 make it easy for comparison. 10 RESPONSE: Thank you for the knowledgeable comment. Normally, we would set the same 11 observation time point for both groups, but the db/db+Rol mice in the Rol supplementation 12 experiment group already showed significant heart systolic dysfunction at 24 w. If we continued 13 to extend the observation time, it may have led to a large number of deaths in the db/db+Rol 14 mice, which would not have been conducive to our data collection and analysis. Therefore, we 15 chose 24 was the experimental endpoint in this part of the experiment. In the atRA 16 supplementation experiment group, no mice showed significant heart systolic dysfunction at 24 17 w. It was not possible to determine the effect of atRA supplementation on heart function in 18 db/db mice at 24 w, so we continued the test until 28 w, when the db/db mice without atRA 19 supplementation showed significant heart systolic dysfunction.